PROJECT SUMMARY: The innate immune system is the dominant immune system found in plants, fungi, insects, and primitive multicellular organisms and comprises the cells and mechanisms that defend the host from infection. In some bacteria, the CRISPR/Cas9 system confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. However, bacteria are also under constant threat from other bacteria, which often attack by employing a Type VI secretion system (T6SS). Bacteria can kill neighboring bacteria via the T6SS-mediated injection of toxic effectors, such as peptidoglycanases (PGases), lipases, nucleases or pore forming toxins. To date, the only known defense mechanism against T6SS-mediated attacks is by the expression of an ?anti-toxin? that recognizes and neutralizes a specific effector. In this project, we hypothesize that some bacteria have developed an ?innate immune system? that allows them to survive the attack of generic T6SS-containing bacteria without expressing specific anti-toxin proteins. We propose that this can be achieved by modifying the peptidoglycan (PG) to make it resistant to degradation by PGase effectors. Peptidoglycan is composed of an N-acetylglucosamine and N-acetylmuramic acid disaccharide with a penta- peptide stem attached, which is then polymerized into glycan strands. These strands are crosslinked together through the peptide stems, forming a meshwork that surrounds the inner membrane. Although the composition of PG seems well-conserved among Gram-negative bacteria, modifications to limit degradation by lysozyme or to elude detection by the host organism have been reported. Our preliminary data indicate that Acinetobacter baumannii (Ab) clinical isolate ATCC 17978 has modified the structure of its peptidoglycan. This modification appears to be biologically relevant as TagX, the PGase that cleaves PG in Acinetobacter to allow the assembly of its own T6SS, is able to degrade Acinetobacter but not Escherichia coli PG. In bacterial competition assays, Acinetobacter species appear to be apex predators, residing at the top of the chain with only a few other species being able to prey on them. We test the hypothesis that PG modifications increase the resistance of Ab to bacterial predators by reducing the activity of bacterial cell wall targeting effectors. Furthermore, we will determine Acinetobacter PG modifications and identify the enzymes involved. Due to the rapid increase of anti- bacterial resistance and a limited number of antibiotics currently being developed, Acinetobacter is quickly becoming a global threat to healthcare and new strategies are needed in order to combat it. Modifications to PG will not only constitute a new paradigm as an immune defense against bacterial attacks but may also provide new targets for the development of antibiotics that interfere with the assembly of the bacterial cell wall.